Phosphorylation of deoxycytidine analogs by cellular enzymes is a prerequisite for the activity of these compounds. We have investigated the kinetic parameters for the phosphorylation of 1-beta-D-arabinofuranosylcytosine (araC) and 2', 2'-difluorodeoxycytidine (dFdC) to their diphosphate forms catalyzed by human UMP-CMP kinase. We cloned the cDNA of this enzyme to enable characterization of the recombinant protein, determine its expression in different tissues, and determine the chromosome location of the gene. We showed that the recombinant UMP-CMP kinase phosphorylated CMP, dCMP, and UMP with highest efficiency and dUMP, AMP, and dAMP with lower efficiency. The monophosphates of araC and dFdC were shown to be phosphorylated with similar efficiency as dCMP and CMP. We further showed, in a combined enzymatic assay, that human deoxycytidine kinase and UMP-CMP kinase together phosphorylated araC, dFdC, and 2',3'-dideoxycytidine to their diphosphate forms. Northern blot analysis showed that the UMP-CMP kinase mRNA was ubiquitously present in human tissues as a 3.9-kb transcript with highest levels in pancreas, skeletal muscle, and liver. The human UMP-CMP kinase gene was localized to chromosome 1p34.1-1p33 by radiation hybrid analysis. We further expressed the UMP-CMP kinase as a fusion protein to the green fluorescent protein in Chinese hamster ovary cells, and showed that the fusion protein was located in the cytosol and nucleus.

Phosphorylation of Deoxycytidine Analog Monophosphates by UMP-CMP Kinase: Molecular Characterization of the Human Enzyme

Human UMP-CMP Kinase 2, a Novel Nucleoside Monophosphate Kinase Localized in Mitochondria

Adenylate kinases have an important role in the synthesis of adenine nucleotides that are required for cellular metabolism. We report the cDNA cloning of a novel 22-kDa human enzyme that is sequence related to the human adenylate kinases and to UMP/CMP kinase of several species. The enzyme was expressed in Escherichia coli and shown to catalyse phosphorylation of AMP and dAMP with ATP as phosphate donor. When GTP was used as phosphate donor, the enzyme phosphorylated AMP, CMP, and to a small extent dCMP. Expression as a fusion protein with the green fluorescent protein showed that the enzyme is located in the cytosol. Northern blot analysis with mRNA from eight different human tissues demonstrated that the enzyme was expressed exclusively in brain, with two mRNA isoforms of 2.4 and 4.0 kb. The gene that encoded the enzyme was localized to chromosome 1p31. Based on the substrate specificity and the sequence similarity with the previously identified human adenylate kinases, we have named this novel enzyme adenylate kinase 5.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1432-1327.1999.00294.x

Identification of a novel human adenylate kinase. cDNA cloning, expression analysis, chromosome localization and characterization of the recombinant protein.

The human adenylate kinase 9 is a nucleoside mono- and diphosphate kinase.

Differences in expression profiles, substrate specificities, kinetic properties and subcellular localization among the AK (adenylate kinase) isoenzymes have been shown to be important for maintaining a proper adenine nucleotide composition for many different cell functions. In the present study, human AK7 was characterized and its substrate specificity, kinetic properties and subcellular localization determined. In addition, a novel member of the human AK family, with two functional domains, was identified and characterized and assigned the name AK8. AK8 is the second known human AK with two complete and active AK domains within its polypeptide chain, a feature that has previously been shown for AK5. The full-length AK8, as well as its two domains AK8p1 and AK8p2, all showed similar AK enzyme activity. AK7, full-length AK8, AK8p1 and AK8p2 phosphorylated AMP, CMP, dAMP and dCMP with ATP as the phosphate donor, and also AMP, CMP and dCMP with GTP as the phosphate donor. Both AK7 and full-length AK8 showed highest affinity for AMP with ATP as the phosphate donor, and proved to be more efficient in AMP phosphorylation as compared with the major cytosolic isoform AK1. Expression of the proteins fused with green fluorescent protein demonstrated a cytosolic localization for both AK7 and AK8.

/pdf/the-characterization-of-human-adenylate-kinases-7-and-8-540sr0jpv6.pdf

The characterization of human adenylate kinases 7 and 8 demonstrates differences in kinetic parameters and structural organization among the family of adenylate kinase isoenzymes.

Jean Lust

Papers

Contributions of anion-exchange thin-layer chromatography of the study of free nucleotides in tissues